Liposomes preformed

In most cases, liposomes are prepared by the film hydration method lipid mixtures are dissolved in organic solvent and then evaporated to dryness. Liposomes are then formed by rehydration of the dried film with the aqueous phase. To encapsulate CyDs within the aqueous core of liposomes, preformed complexes are generally dissolved in the aqueous rehydration phase. Another technique is to intro-... 

A method resulting in improved encapsulation of aqueous phase by MLV is the so-called dehydration-rehydration procedure (Kirby and Gregoriadis, 1984 Shew and Deamer, 1985). The lipid (usually preformed liposomes) is dried (by either lyophilization or evaporation) in the presence of the aqueous solute to be entrapped, thus forming a mixed film with solute trapped between layers. Subsequent gradual rehydration with a minimum of aqueous phase leads to the formation of MLV with a high entrapment of the aqueous solutes added. 

Amphipathic Weak Base Loading into Preformed Liposomes Having a Transmembrane Ammonium Ion Gradient From the Bench to Approved Doxil... 

Numerous methods have been developed for attaching ligands to the surface of liposomes for reviews, see Refs. (3,6 ). For peptides, they fall into two major categories (i) covalent coupling of the peptides to preformed liposomes that contain functionalized hydrophobic anchors such as, e.g., derivatives of phos-phatidylethanolamine (PE) or (ii) incorporation of lipopeptides, obtained by conjugation of peptides to hydrophobic anchors (fatty acids, phospholipids), into liposomes either during the preparation of the vesicles or by postinsertion into preformed vesicles. In this section, we will briefly discuss these techniques and focus on the ones we have been using in our own work. 

Figure 1 Conjugation reactions for coupling peptides to the surface of preformed liposomes. Functionalized lipophilic anchors were incorporated into liposomes and reacted with the peptides in aqueous media. Reactive endgroup functions (A) maleimide (B) bromo- or iodoacetyl (C) 2-pyridyldithio (D) carboxylic acid (E) />-nitrophenyl carbonate and (F) hydrazide. Abbreviations EDC, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide NHS, 7V-hydroxysuccinimide.

Several alternative techniques can be exploited to conjugate peptides to preformed liposomes. They all involve hydrophobic anchors incorporated into the bilayers of vesicles that are able to react with modified or unmodified peptides. Their principles will be listed below, accompanied by pertinent references. 

The two examples from our work we are going to describe below are the design and study of liposomal diepitope constructs combining either (i) B and T-helper (Th) peptide epitopes, which induced particularly powerful humoral responses (21) (Fig. 3) or (ii) CTL and Th epitopes, which provided a powerful antitumor vaccine (74) (Fig. 4). For the production of these constructs we have conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactive functionalized phospholipids and/or PamaCys lipopeptide anchors (Fig. 2). To that end, we have developed strategies that give, in aqueous media, high... 

The preformed vesicle (PFV) approach involves incubation of liposomes containing a cationic lipid and a PEG coating with polynucleotides in the presence of ethanol. Typically, LUV composed of distearoyl-phosphatidyl-choline (DSPC), cholesterol (Choi), l-0-(2 -(oi-methoxy-polyethylene-glycol)... 

In summary, the preformed vesicle approach and detergent dialysis procedure have enabled development of nucleic acid-based therapeutics with clinical utility. Further applications of these liposomal systems with new nucleic acid-based therapeutics such as small interfering RNA for gene silencing are being developed and have demonstrated promising results (28). 

The BMEDA method shares some of the same features as the HMPAO method such as good in vitro and in vivo stability with a variety of preformed liposome formulations, and the need for coencapsulation of glutathione. In addition, an advantage of the BMEDA-labeling method is that it can also be used for labeling liposomes with therapeutic rhenium radionuclides (22). Currently, for the BMEDA method, there is no commercially available kit. Also the BMEDA chemical is not currently commercially available and must be synthesized. 

Hnatowich DJ, Friedman B, Clancy B, Novak M. Labeling of preformed liposomes with Ga-67 and Tc-99m by chelation. J Nucl Med 1981 22 810. 

The weakly immunogenic protamine sulfate USP (1) condenses DNA to form a toroid structure of super-coiled DNA about 50 nm in diameter (2). The DNA in this form or in the preformed LPDI complex cannot be displaced from the protamine by polycations such as spermidine and histones or by other nucleic acids like genomic DNA (2). DNA in this toroid structure is transcriptionally inactive and this conformation allows for protection of DNA from enzymatic degradation by nucleases and other environmental assaults such as mechanical stress (1,2). After the liposome surrounds the toroid, the resulting homogenous LPDI nanoparticles are slightly less than... 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

Figure 2.10. Schematic diagram of coupling of a thiolated antibody to a linker lipid (maleimide-PEG-phospholipid) which is part of a preformed liposome. The resulting thioether bond is meta-bolically stable. The strategy shown here was used to synthesize OX26-immunoliposomes [111].

Figure 11.7 Loading of a weakly basic drug into preformed liposomes by pH gradient.

Insertion of scVEGF-Lipid Conjugate into Preformed Liposomes... 

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